Unsymmetrical fluorescein derivatives

ABSTRACT

Unsymmetrically substituted 6-hydroxy-3H-xanthen-3-ones for use as fluorescers or quenchers, particularly conjugated to provide reagents in competitive protein binding assays. Substituents are aliphatic groups bonded to an annular carbon atom through carbon or oxygen.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Fluorescent compounds find a wide variety of applications. They find usein fluorescent immunoassays, histochemical staining, displays, inks, andthe like. Of particular interest for the subject invention is the use ofantigenic conjugates (includes receptor conjugates) with fluorescentcompounds to be used in the determination of a variety of ligands, bothantigens and receptors. A substantial proportion of the ligands areassayed in physiological fluids, such as serum, where the serum canprovide substantial background fluorescence. One way to diminish thebackground fluorescence resulting from naturally present fluorescers isto provide a fluorescent compound which absorbs at relatively longwavelengths. The compound should desirably have a large Stokes shift, bestable under conditions of the assay, be relatively free of non-specificinterference, both from materials in solution and the compound to whichthe fluorescer is conjugated, and provide high quantum yields. Inaddition, for certain applications, it is desirable that the fluorescerbe coupled with a quencher molecule, that is a molecule which is capableof absorbing the energy of the fluorescer in the excited state, whenwithin a predetermined distance, so that the fluorescer does notfluoresce.

2. Description of the Prior Art

A large number of fluorescein derivatives have been reported in theliterature. The following are believed to be the most exemplary inrelation to the subject invention and are reported in conjunction withthe Chemical Abstracts citation. The numbering is based on the parentmolecule 3',6'-dihydroxyspiro[isobenzofuran-1(3H),9'-(9H)xanthen]-3-one.

Asymmetrical fluorescein derivatives without functionalities forattaching to other molecules have been reported in the literature. Thefollowing is not intended to be exhaustive of such compounds. TheChemical Abstracts reference is indicated. 2',4'-dichloro-7'-bromo,2',7'-dichloro-4'-bromo, 2,7'-dibromo-4'-chloro,2'-bromo-4',7'-dichloro, 2',4'-dibromo-7'-chloro, C.A. 62, 13116C;2',7'-dibromo-4'-hydroxy, C.A. 61, 7407d; 2',4',7'-tribromo, C.A. 53,9573h, 65, 4328a; 2',4',7'-trichloro, C.A. 55, 12393f, 63, 6954f;2',4',5'-triiodo, C.A. 57, 11794b; 2',4',5'-tribromo, C.A. 53, 9573h;2',4',5'-trichloro, C.A. 55, 12393f, 63, 6954f; 4',5'-dibromo-2'-iodo,C.A. 49, 254e; 4',5'-dinitro-2'-hydroxymercuri, C.A. 51, 313i;2',5'-dichloro, 2',4'-dichloro, 4'-chloro, 2' -chloro, C.A. 55, 12393f,63 6954f; 2'-bromo-5'-chloro, C.A. 62, 13116c; 2',5'-dibromo, C.A. 53,9573g, 71, 8201e; 4'-[(4-sulfamoyl-O-tolyl)azo],4'-[(p-[2-thiazolylsulfamoyl]phenyl)azo], 4'-[(p-sulfamoylphenyl)azo],4'-[(6-sulfamoyl-m-tolyl)azo], 4'-[(p-[2-pyridylsulfamoyl]phenyl)azo],C.A. 72, 91456d; 4'-iodo, C.A. 57, 11794c; 4'-chloro, C.A. 63, 6954f,70, 2018n; 4'-bromo, C.A. 62, 5249b, 71, 8201c; 2'-pentenyl, 2'-allyl,C.A. 31, 1388; 2'-benzyl, C.A. 74, 45540v; 2'-bromo, C.A. 53, 9573h, 62,5249c, 71, 8201c.

Symmetrical fluorescein derivatives, having alkyl or oxy substituentsmay be found in co-pending applications Ser. No. 73,158, filed Sept. 7,1979 and 73,163, filed Sept. 7, 1979, now U.S. Pat. Nos. 4,318,846 and4,351,760, respectively.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Unsymmetrical fluorescein derivatives are provided having high Stokesshifts and high quantum yields. The compounds can be used whereinterference is observed due to the presence of endogenous fluorescershaving absorption at longer wavelengths, such as found in serum. Thecompounds can also be used as conjugates to make other compounds orsubstrates fluorescent, as labels in histochemistry, cytology, anddiagnostic assays. Depending upon the substituents employed on thefluorescein, the compound can be fluorescent or be non-fluorescent andcapable of acting as a quencher.

The subject invention concerns fluorescent compounds, which are analogsof fluorescein, being particularly1,8-unsubstituted-9-substituted-6-hydroxy-3H-xanthen-3-ones, having onealiphatic substituent at any of the remaining positions, where thealiphatic substituent is separated from the annular carbon atom by from0 to 1 oxygen atom. Normally present will be a functionality,particularly a non-oxo-carbonyl functionality, for conjugation to amember of an immunological pair, referred to as a ligand and receptor.For convenience, the member of the immunological pair will beabbreviated as "mip". The conjugates to the mip find particular use asreagents in assays for determining a mip.

The fluorescent precursors will have at least about 14 carbon atoms,usually about 20 carbon atoms, and usually not more than about 40 carbonatoms, usually having from about 21 to 35 carbon atoms. Preferably thereis at least one, usually two chlorine atoms at other than the1,8-positions and there may be as many as 7 chlorine atoms. In additionto chlorine, the only other heteroatoms are fluorine, bromine, andiodine, chalcogen, particularly oxygen and sulfur, and nitrogen, therebeing at least 4 heteroatoms and usually not more than 20 heteroatoms,more usually not more than about 16 heteroatoms and preferably not morethan about 12 heteroatoms. Of the heteroatoms other than chlorine, therewill be at least 3 oxygen atoms, more usually at least 5 oxygen atoms,and other than the oxygen atoms, which are part of the xanthenechromophore, are oxygen atoms as non-oxo-carbonyl or oxy, particularlyacid, ester or ether (normally bonded solely to carbon and hydrogen);sulfur is normally present as sulfonyl, thioether or mercapto; whilenitrogen is normally present as amino or amido (bonded solely to carbonand hydrogen).

The fluorescent compounds are further characterized by having absorptionmaxima in 0.5 M phosphate buffer pH 8 of at least about 490 nm, usuallyat least about 500 nm, an extinction coefficient in the same medium ofat least about 65,000 M⁻¹ cm⁻¹,, more usually at least 70,000 and aStokes shift in the same medium of at least about 10 nm, more usually atleast about 12 nm.

The 9-substituted-xanthenes of this invention will for the most parthave the following formula: ##STR1## wherein: one of the γ's will be ofthe formula --(O).sub.ε ρ and the other is δ;

wherein:

O stands for its normal meaning of oxygen;

ε is zero or one;

ρ is an aliphatic group, normally aliphatic hydrocarbon or substitutedaliphatic hydrocarbon, usually monosubstituted, saturated orunsaturated, branched or straight chain, particularly alkyl orcarboxyalkyl of from 1 to 12, usually 1 to 6, more usually 1 to 4 carbonatoms, and having from 0-1 carboxyl groups;

δ is hydrogen, or halogen of atomic number 17 to 80, the δ's may be thesame or different;

L is a bond or divalent radical, usually an organic radical, of at leastone carbon atom and not more than 20, usually not more than 16, moreusually not more than 10 carbon atoms, normally having an aliphatic oraromatic hydrocarbon chain or combination thereof, wherein the aliphaticchain is usually of from about 2 to 6 carbon atoms, and the aromaticchain of from about 6 to 12, usually 6 to 10 carbon atoms; L normallyhas from 0 to 4, when aromatic, usually 1 to 4, more usually 2 to 4substituents, wherein the substituents may be halo, particularly chloro,non-oxo-carbonyl, thio, including inert sulfur acids, esters and amides,amino, particularly tert-amino or amido, and oxy; wherein thesubstituents are normally of from 0 to 4 carbon atoms, there being atleast two carbon atoms between heteroatoms bonded to saturated carbonatoms; when L is a benzene ring, 4 will be at other than the orthoposition to the xanthenone.

α may be taken together with Ψ to define an active group capable offorming a stable covalent bond with carbon, nitrogen or oxygen or is anorganic compound, usually a member of a specific binding pair, either aligand or receptor; the covalent bond formed between Ψ and α normallyinvolves an amido, methylene, sec-amino, ether, thio, or azo link, withthe proviso that when ρ has a carboxyl substituent, α may be takentogether with Ψ to define H, oxy, or halo of atomic number 9 to 80;

Ψ is a group terminating in a heteroatom containing functionality whennot taken together with α, wherein the terminal heteroatom containingfunctionality may be bonded directly to a carbon atom of L or through anoligomer of from 1 to 4 units, each unit of 1 to 4, usually 2 to 4carbon atoms, which units are amino acids of from 2 to 4 carbon atoms,alkyleneamino or alkyleneoxy groups; the terminal functionality isnormally oxo, including oxo-carbonyl and non-oxo-carbonyl; amino; oxy;thio; or active halogen; particularly non-oxo-carbonyl; and v is 1 whenΨ is taken together with α to form an active group, and is otherwise onthe average of at least 1 and not more than the molecular weight of αdivided by 500, usually divided by 5,000.

When ε is 0, that is, one of the γ's is an aliphatic group, one of theδ's may be an aliphatic group coming within the definition of ρ, wherethe two aliphatic groups are asymmetrically positioned. The numericalvalue of the positions to which the aliphatic groups are bonded will addup to other than nine. Therefore, the dialiphatic substitutedxanthenones would be 2,4-, 2,5- and 4,7-substituted.

Desirably, there are from 0 to 6, more usually from 1 to 6 chlorosubstituents on the fluorescent group (in the brackets), bonded at otherthan the 1,8-positions of the xanthenone. Also, while there may be onlyone oxy substituent at any of the positions, 2,4,5, and 7, there may beup to two aliphatic substituents where ε is 0, and the sum of thepositions of the alkyl substituents adds up to other than 9.

For the most part, the compounds of this invention will have a 9-phenylsubstituent and will be of the following formula: ##STR2## wherein: oneof the Gs is of the formula --(O)_(e) R:

wherein:

e is 0 or 1;

R is an aliphatic group of from 1 to 6, usually 1 to 4, more usually 1to 3 carbon atoms, which may be substituted or unsubstituted,aliphatically saturated or unsaturated, usually saturated, butparticularly alkyl or carboxyalkyl of from 1 to 4, usually 1 to 3 carbonatoms;

when other than of the formula --(O)_(e) R, G will come within thedefinition of D;

Z is carboxy or sulphonoxy;

D is hydrogen or halogen of atomic number 17 to 80, usually 17 to 35;

W is a bond or divalent organic radical having from 0 to 16, either 0 orfrom 1 to 10, more usually 1 to 8, preferably 1 to 6 carbon atoms andfrom 0 to 8, usually 2 to 8, more usually 2 to 6 heteroatoms, which arechalcogen, (oxygen and sulfur) or nitrogen; wherein chalcogen is presentbonded solely to carbon (oxy or oxo) and nitrogen is present bondedsolely to carbon and hydrogen (amino and amido); carbon is normallyaromatic or aliphatic, particularly free of aliphatic unsaturation,having from 0 to 2 sites of ethylenic unsaturation, preferablysaturated; W is conveniently a monomer or oligomer of units of from 1 to4 carbon atoms e.g. alkylene, amino acid, oxyalkylene, aminoalkylene,etc;

Y may be taken together with A to form an active functionality capableof forming a covalent bond with a heterofunctionality, such as amino,hydroxy, or mercapto; that is, with those functionalities present on A,when A is other than taken together with Y. Y and A taken together arefunctionalities such as oxo, either oxo- or non-oxo-carbonyl, oxy, thio,amino, active halo, active olefin, inorganic acyl group, e.g. sulfonyl,etc., or when not taken together with A, Y acts as a linkingfunctionality, being either methylene or heteroatom containing, e.g.,non-oxo-carbonyl, thiourea, sulfonyl, oxy, imino, etc.; when W and Y aretaken together to define carboxy, usually non-oxo-carbonyl WY will be atother than the ortho position;

A, when not taken together with Y, is a member of a specific bindingpair, which is a ligand or receptor, wherein the ligand may be haptenicor antigenic, normally being of from about 125 molecular weight to aindefinite upper limit. Although for the most part, most ligands will beunder 10 million molecular weight, more usually under 2 millionmolecular weight, with varying ranges depending upon the nature of theligand or receptor, of particular interest are poly(amino acid) ligandsor receptors of from about 1,000 to 500,000 molecular weight;

X is halo, usually of atomic number 9 to 80, more usually 9 to 35,particularly chloro;

m is 0 to 3, more usually 1 to 2;

p is 0 to 3, with the sum of m and p being not greater than 4; and,

n will be 1 when Y and A are taken together and will otherwise be on theaverage 1 to the molecular weight of A divided by 500, more usuallydivided by 1,000, and more frequently divided by 2,000, wherein withspecific binding pair members over 600,000 molecular weight, it willnormally be not greater than A divided by 5,000. Furthermore, either theconjugate or the fluorescer precursor may be bonded to a support of atleast about 10,000 molecular weight and up to an indefinite molecularweight.

A preferred group of compounds have the following formula: ##STR3##wherein: one of G' or G", preferably G' is --(O)₃ 'R';

wherein:

e' is 0 or 1, preferably 1 and

R' is alkyl of from 1 to 6, more usually of from 1 to 3 carbon atoms;

the other of G' and G" will come within the definition of D';

X' and Z' come within the definitions of X and Z, respectively;

D' is hydrogen or halogen of atomic number 17 to 80, usually 17 to 35,and preferably chloro;

Y' may be taken together with A' to define a non-oxo-carbonyl group orwhen not taken together with A' is a non-oxo-carbonyl linking group toA', usually forming an amide link; when taken together with A' to definecarboxy, --W'Y' will be at other than the ortho position;

W' is a bond or linking group, usually aliphatic, of from 1 to 12,usually 1 to 8 atoms other than hydrogen, which are carbon, nitrogen,oxygen or sulfur, preferably carbon, nitrogen, and oxygen, there beingfrom 0 to 8 carbon atoms and 0 to 8 heteroatoms, preferably 0 to 4heteroatoms, with then number of carbon atoms and hetero atoms being atleast 1, wherein nitrogen will be bonded solely to hydrogen and carbonand will be either amino or amido, oxygen and sulfur will be bondedsolely to carbon as oxy(thio) or oxo(thiono) and carbon is normallyaliphatic, usually free of aliphatic unsaturation, generally having from0 to 1 site of ethylenic unsaturation; W' may be alkylene, alkenylene,oxyalkylene, or oxoalkylene of from 1 to 8, usually 1 to 4 carbon atoms,imino (NH), N-formyl amino acid or N-formyl poly(amino acid) e.g.glycine or polyglycine, there being from 1 to 4, usually 1 to 2, aminoacids of from 2 to 4 carbon atoms, with the terminal carboxy being Y';

n' is 1 when Y' and A' are taken together and otherwise is on theaverage at least 1 to the molecular weight of A' divided by 500, usuallydivided by 1,000, and more usually divided by 2,000, and when A' is over500,000 molecular weight, more usually divided by 5,000;

m' is 0 to 2, usually 0 to 1;

p' is 0 to 3, more usually 0 to 2, the sum of m' and p' being notgreater than 4;

there generally being not more than three carboxyl groups, more usuallynot more than about two carboxyl groups in total, and there being fromabout 0 to 6 chloro groups, more usually from about 0 to 5 chlorogroups, and preferably from about 2 to 5 chloro groups; and

A' when not taken together with Y' is a member of a specific bindingpair, a ligand or receptor, wherein the ligand may be haptenic orantigenic, where haptenic ligands will include compounds of interestsuch as drugs, hormones, pollutants, processing compounds, agriculturalchemicals, metabolites, and the like; antigens will primarily beproteins, polysaccharides, or nucleic acids, individually or incombination with each other, or other materials involving combinationsof compounds, such as cells, viruses, phage, or the like. The haptenswill normally be from about 125 to 2,000 daltons, more usually to 1,000daltons, while the antigens will normally be from about 2,000, moreusually 5,000 daltons up to an indefinite molecular weight, usually notexceeding 10 million daltons, more usually not exceeding 2 millionsdaltons.

Preferably, three of the 2,4,5, and 7 positions will be unsubstituted orchloro substituted.

In some instances, it may be desirable to have the fluorescent compoundor the conjugate of the fluorescent compound with the ligand orreceptor, bonded to a support, where the linkage may be derived fromeither the fluorescent compound or the ligand or receptor compound. Inthis situation, the linking group may be any conventional functionalitywhich is present on the fluorescent compound or the ligand or receptor,or a functionality which may be introduced onto any of these compounds.

The supports will include macromolecular supports of at least about10,000 molecular weight, which may be naturally occurring or synthetic,having a plurality of functionalities for linking e.g. carboxy, hydroxy,or amino, usually being a polymer, such as a polysaccharide or anaddition polymer.

Quite obviously, the compounds of the subject invention can be modifiedso as not to be within the above formulas, without significantlyaffecting the properties of the compounds. For example, one or more ofthe acidic anionic groups can be esterified or amidified or alkyl groupscan be substituted on the phenyl as well as other groups, such as cyano,nitro or the like. However, these changes will in most cases requireadditional synthetic steps which are not warrented by the degree ofenhancement, if any, in the spectroscopic or chemical properties of theresulting product.

Turning now to a consideration of the individual components of thesubject compositions, the fluorescein derivatives will be consideredfirst. The following is a list of illustrative fluorescein derivativescoming within the scope of the subject invention.

TABLE I

2-methyl-4,5-dichloro-9-(2',4',5'-tricarboxyphenyl)-6-hydroxy-3H-xanthen-3-one

2-ethyl-4,5-dichloro-9-(2',4',5'-tricarboxy-3',6'-dichlorophenyl)-6-hydroxy-3H-xanthen-3-one

2-hexyl-9-(2',4',5'-tricarboxyphenyl)-6-hydroxy-3H-xanthen-3-one

2-methyl-4,5-dichloro-9-(2'-carboxy-4'-isothiocyanato-3',5'-dichlorophenyl)-6-hydroxy-3H-xanthen-3-one

2-methyl-9-(2'-carboxy-4'-isocyanato-3',5',6'-trichlorophenyl)-6-hydroxy-3H-xanthen-3-one

2-methyl-9-(4'-carboxamidoacetamidoglycylglycine-5'-carboxylphenyl)-6-hydroxy-3H-xanthen-3-one

2-propoxy-9-(4',5',-dicarboxy-2',3',6'-trichlorophenyl)-6-hydroxy-3H-xanthen-3-one

2-ethoxy-4,5-dichloro-9-(3',4'-dicarboxyphenyl)-6-hydroxy-3H-xanthen-3-one

2-ethyl-9-(2'-carboxy-4'-amino-3',5'-dichlorphenyl)-6-hydroxy-3H-xanthen-3-one

4-ethyl-9-(2'-carboxy-4'-mercaptophenyl)-6-hydroxy-3H-xanthen-3-one

4-ethoxy-9-(2'-carboxy-4'-carboxymethylphenyl)-6-hydroxy-3H-xanthen-3-one

2-ethoxy-9-(2'-carboxy-4'-(4"-carboxybutyl)-phenyl)-6-hydroxy-3H-xanthen-3-one

2-methoxy-4,5-dichloro-9-(2',4'-dicarboxy-5'-(carboxyamidomethylene)phenyl)-6-hydroxy-3H-xanthen-3-one

2 methyl-4,5-dichloro-9(3'-carboxypropyl)-6-hydroxy-3H-xanthen-3-one.

As indicated previously, the fluorescein derivatives of the subjectinvention will be conjugated with ligands and/or supports. The followingis a description of the applicable ligands.

Analyte

The ligand analytes of this invention are characterized by beingmonoepitopic or polyepitopic. The polyepitopic ligand analytes willnormally be poly(amino acids) i.e. polypeptides and proteins,polysaccharides, nucleic acids, and combinations thereof. Suchcombinations of assemblages include bacteria, viruses, chromosomes,genes, mitochondria, nuclei, cell membranes, and the like.

For the most part, the polyepitopic ligand analytes employed in thesubject invention will have a molecular weight of at least about 5,000,more usually at least about 10,000. In the poly(amino acid) category,the poly(amino acids) of interest will generally be from about 5,000 to5,000,000 molecular weight, more usually from about 20,000 to 1,000,000molecular weight; among the hormones of interest, the molecular weightswill usually range from about 5,000 to 60,000 molecular weight.

The wide variety of proteins may be considered as to the family ofproteins having similar structural features, proteins having particularbiological functions, proteins related to specific microorganisms,particularly disease causing microorganisms, etc.

The following are classes of proteins related by structure:

protamines

histones

albumins

globulins

scleroproteins

phosphoproteins

mucoproteins

chromoproteins

lipoproteins

nucleoproteins

glycoproteins

proteoglycans

unclassified proteins, e.g. somatotropin, prolactin, insulin, pepsin

A number of proteins found in the human plasma are important clinicallyand include:

Prealbumin

Albumin

α₁ -Lipoprotein

α₁ -Acid glycoprotein

α₁ -Antitrypsin

α₁ -Glycoprotein

Transcortin

4.6S -Postalbumin

Tryptophan-poor

α₁ -glycoprotein

α₁ X-Glycoprotein

Thyroxin-binding globulin

Inter-α-trypsin-inhibitor

Gc-globulin

(Gc 1-1)

(Gc 2-1)

(Gc 2-2)

Haptoglobin

(HP 1-1)

(HP 2-1)

(Hp 2-2)

Ceruloplasmin

Cholinesterase

α₂ -Lipoprotein(s)

Myoglobin

C-Reactive Protein

α₂ -Macroglobulin

α₂ -HS-glycoprotein

Zn-α₂ -glycoprotein

α₂ -Neuramino-glycoprotein

Erythropoietin

β-lipoprotein

Transferrin

Hemopexin

Fibrinogen

Plasminogen

β₂ -glycoprotein I

β₂ -glycoprotein II

Immunoglobulin G

(IgG) of γG-globulin

Mol. formula:

γ₂ κ₂ or γ₂ λ₂

Immunoglobulin A (IgA)

or γA-globulin

Mol. formula:

(α₂ κ₂)^(n) or (α₂ λ₂)^(n)

Immunoglobulin M

(IgM) or γM-globulin

Mol. formula:

(μ₂ κ₂)⁵ or (μ₂ λ₂)⁵

Immunoglobulin D(IgD)

or γD-Globulin (γD)

Mol. formula:

δ₂ κ₂) or δ₂ λ₂)

Immunoglobulin E (IgE)

or γE-Globulin (γE)

Mol. formula:

(ε₂ κ₂) or (ε₂ λ₂)

Free κ and λ light chains

Complement factors:

C'1

C'1q

C'1r

C'1s

C'2

C'3

β₁ A

α₂ D

C'4

C'5

C'6

C'7

C'8

C'9

Important blood clotting factors include:

    ______________________________________                                        BLOOD CLOTTING FACTORS                                                        International designation                                                                       Name                                                        ______________________________________                                        I                 Fibrinogen                                                  II                Prothrombin                                                 IIa               Thrombin                                                    III               Tissue thromboplastin                                       V and VI          Proaccelerin, accelerator                                                     globulin                                                    VII               Proconvertin                                                VIII              Antihemophilic globulin                                                       (AHG)                                                       IX                Christmas factor,                                                             plasma thromboplastin                                                         component (PTC)                                             X                 Stuart-Prower factor,                                                         autoprothrombin III                                         XI                Plasma thromboplastin                                                         antecedent (PTA)                                            XII               Hagemann factor                                             XIII              Fibrin-stabilizing factor                                   ______________________________________                                    

Important protein hormones include:

Peptide and Protein Hormones

Parathyroid hormone (parathromone)

Thyrocalcitonin

Insulin

Glucagon

Relaxin

Erythropoietin

Melanotropin (melanocyte-stimulating hormone; intermedin)

Somatotropin (growth hormone)

Corticotropin (adrenocorticotropic hormone)

Thyrotropin

Follicle-stimulating hormone

Luteinizing hormone (interstitial cell-stimulating hormone)

Luteomammotropic hormone (luteotropin, prolactin)

Gonadotropin (chorionic gonadotropin)

Tissue Hormones

Secretin

Gastrin

Angiotensin I and II

Bradykinin

Human placental lactogen

Peptide Hormones from the Neurohypophysis

Oxytocin

Vasopressin

Releasing factors (RF) CRF, LRF, TRF, Somatotropin-RF, GRF, FSH-RF, PIF,MIF

Other polymeric materials of interest are mucopolysaccharides andpolysaccharides.

Illustrative antigenic polysaccharides derived from microorganisms areas follows:

    ______________________________________                                        Species of Microorganisms                                                                        Hemosensitin Found in                                      ______________________________________                                        Streptococcus pyogenes                                                                           Polysaccharide                                             Diplococcus pneumoniae                                                                           Polysaccharide                                             Neisseria meningitidis                                                                           Polysaccharide                                             Neisseria gonorrheae                                                                             Polysaccharide                                             Corynebacterium diphtheriae                                                                      Polysaccharide                                             Actinobacillus mallei;                                                                           Crude extract                                              Actinobacillus whitemori                                                      Francisella tularensis                                                                           Lipopolysac-                                                                  charide                                                                       Polysaccharide                                             Pasteurella pestis                                                            Pasteurella pestis Polysaccharide                                             Pasteurella multocida                                                                            Capsular antigen                                           Brucella abortus   Crude extract                                              Haemophilus influenzae                                                                           Polysaccharide                                             Haemophilus pertussis                                                                            Crude                                                      Treponema reiteri  Polysaccharide                                             Veillonella        Lipopolysac-                                                                  charide                                                    Erysipelothrix     Polysaccharide                                             Listeria monocytogenes                                                                           Polysaccharide                                             Chromobacterium    Lipopolysac-                                                                  charide                                                    Mycobacterium tuberculosis                                                                       Saline extract of                                                             90% phenol                                                                    extracted                                                                     mycobacteria                                                                  and polysac-                                                                  charide                                                                       fraction of                                                                   cells and                                                                     turberculin                                                Klebsiella aerogenes                                                                             Polysaccharide                                             Klebsiella cloacae Polysaccharide                                             Salmonella typhosa Lipopolysac-                                                                  charide,                                                                      Polysaccharide                                             Salmonella typhi-murium;                                                                         Polysaccharide                                             Salmonella derby                                                              Salmonella pullorum                                                           Shigella dysenteriae                                                                             Polysaccharide                                             Shigella flexneri                                                             Shigella sonnei    Crude, Poly-                                                                  saccharide                                                 Rickettsiae        Crude extract                                              Candida albicans   Polysaccharide                                             Entamoeba histolytica                                                                            Crude extract                                              ______________________________________                                    

The microorganisms which are assayed may be intact, lysed, ground orotherwise fragmented, and the resulting composition or portion, e.g. byextraction, assayed. Microorganisms of interest include:

Corynebacteria

Corynebacterium diptheriae

Pneumococci

Diplococcus pneumoniae

Streptococci

Streptococcus pyogenes

Streptococcus salivarus

Staphylococci

Staphylococcus aureus

Staphylococcus albus

Neisseriae

Neisseria meningitidis

Neisseria gonorrheae

Enterobacteriaciae

The coliform bacteria

Escherichia coli

Aerobacter aerogenes

Klebsiella pneumoniae

The Salmonellae

Salmonella typhosa

Salmonella choleraesuis

Salmonella typhimurium

The Shigellae

Shigella dysenteriae

Shigella schmitzii

Shigella arabinotarda

Shigella flexneri

Shigella boydii

Shigella Sonnei

Other enteric bacilli

Proteus species

Proteus vulgaris

Proteus mirabilis

Proteus morgani

Pseudomonas aeruginosa

Alcaligenes faecalis

Vibrio cholerae

Hemophilus-Bordetella group

Hemophilus influenzae, H. ducreyi, H. hemophilus, H. aegypticus, H.parainfluenzae,

Bordetella pertussis

Pasteurellae

Pasteurella pestis

Pasteurella tulareusis

Brucellae

Brucella melitensis

Brucella abortus

Brucella suis

Aerobic Spore-forming Bacilli

Bacillus anthracis

Bacillus subtilis

Bacillus megaterium

Bacillus cereus

Anaerobic Spore-forming Bacilli

Clostridium botulinum

Clostridium tetani

Clostridium perfringens

Clostridium novyi

Clostridium septicum

Clostridium histolyticum

Clostridium tertium

Clostridium bifermentans

Clostridium sporogenes

Mycobacteria

Mycobacterium tuberculosis hominis

Mycobacterium bovis

Mycobacterium avium

Mycobacterium leprae

Mycobacterium paratuberculosis

Actinomycetes (fungus-like bacteria)

Actinomyces israelii

Actinomyces bovis

Actinomyces naeslundii

Nocardia asteroides

Nocardia brasiliensis

The Spirochetes

Treponema pallidum

Treponema pertenue

Spirillum minus

Streptobacillus moniliformis

Treponema carateum

Borrelia recurrentis

Leptospira icterohemorrhagiae

Leptospira canicola

Mycoplasmas

Mycoplasma pneumoniae

Other pathogens

Listeria monocytogenes

Erysipelothrix rhusiopathiae

Streptobacillus moniliformis

Donvania granulomatis

Bartonella bacilliformis

Rickettsiae (bacteria-like parasites)

Rickettsia prowazekii

Rickettsia mooseri

Rickettsia rickettsii

Rickettsia conori

Rickettsia australis

Rickettsia sibiricus

Rickettsia akari

Rickettsia tsutsugamushi

Rickettsia burnetii

Rickettsia quintana

Chlamydia (unclassifiable parasites bacterial/viral)

Chlamydia agents (naming uncertain)

Fungi

Cryptococcus neoformans

Blastomyces dermatidis

Histoplasma capsulatum

Coccidioides immitis

Paracoccidioides brasiliensis

Candida albicans

Aspergillus fumigatus

Mucor corymbifer (Absidia corymbifera)

Phycomycetes

Rhizopus oryzae

Rhizopus arrhizus

Rhizopus nigricans

Sporotrichum schenkii

Fonsecaea pedrosoi

Fonsecaea compacta

Fonsecaea dermatidis

Cladosporium carrionii

Phialophora verrucosa

Aspergillus nidulans

Madurella mycetomi

Madurella grisea

Allescheria boydii

Phialosphora jeanselmei

Microsporum gypseum

Trichophyton mentagrophytes

Keratinomyces ajelloi

Microsporum canis

Trichophyton rubrum

Microsporum andouini

Viruses

Adenoviruses

Herpes Viruses

Herpes simplex

Varicella (Chicken pox)

Herpes Zoster (Shingles)

Virus B

Cytomegalovirus

Pox Viruses

Variola (smallpox)

Vaccinia

Poxvirus bovis

Paravaccinia

Molluscum contagiosum

Picornaviruses

Poliovirus

Coxsackievirus

Echoviruses

Rhinoviruses

Myxoviruses

Influenza (A, B, and C)

Parainfluenza (1-4)

Mumps Virus

Newcastle Disease Virus

Measles Virus

Rinderpest Virus

Canine Distempeer Virus

Respiratory Syncytial Virus

Rubella Virus

Arboviruses

Eastern Equine Eucephalitis Virus

Western Equine Eucephalitis Virus

Sindbis Virus

Chikugunya Virus

Semliki Forest Virus

Mayora Virus

St. Louis Encephalitis Virus

California Encephalitis Virus

Colorado Tick Fever Virus

Yellow Fever Virus

Dengue Virus

Reoviruses

Reovirus Types 1-3

Hepatitis

Hepatitis A Virus

Hepatitis B Virus

Tumor Viruses

Rauscher Leukemia Virus

Gross Virus

Maloney Leukemia Virus

The monoepitopic ligand analytes will generally be from about 100 to2,000 molecular weight, more usually from 125 to 1,000 molecular weight.The analytes of interest include drugs, metabolites, pesticides,pollutants, and the like. Included among drugs of interest are thealkaloids. Among the alkaloids are morphine alkaloids, which includesmorphine, codeine, heroin, dextromethorphan, their derivatives andmetabolites; cocaine alkaloids, which includes cocaine and benzoylecgonine, their derivatives and metabolites; ergot alkaloids, whichincludes the diethylamide of lysergic acid; steroid alkaloids; iminazoylalkaloids; quinazoline alkaloids, isoquinoline alkaloids; quinolinealkaloids; which includes quinine and quinidine; diterpene alkaloids;their derivatives and metabolites.

The next group of drugs includes steroids, which includes the estrogens,gestogens, androgens, andrenocortical steroids, bile acids, cardiotonicglycosides and aglycones, which includes digoxin and digoxigenin,saponins and sapogenins, their derivatives and metabolites. Alsoincluded are the steriod mimetic substances, such as diethylstilbestrol.

The next group of drugs is lactams having from 5 to 6 annular members,which include the barbiturates, e.g. phenobarbital and secobarbital,diphenylhydantonin, primidone, ethosuximide, and their metabolites.

The next group of drugs is aminoalkylbenzenes, with alkyl of from 2 to 3carbon atoms, which includes the amphetamines, catecholamines, whichincludes ephedrine, L-dopa, epinephrine, narceine, papaverine, theirmetabolites.

The next group of drugs is aminoalkylbenzenes, with alkyl of from 2 to 3carbon atoms, which includes ephedrine, L-dopa, epinephrine, narceine,papaverine, their metabolites and derivatives.

The next group of drugs is benzheterocyclics which include oxazepam,chlorpromazine, tegretol, imipramine, their derivatives and metabolites,the heterocyclic rings being azepines, diazepines and phenothiazines.

The next group of drugs is purines, which includes theophylline,caffeine, their metabolites and derivatives.

The next group of drugs includes those derived from marijuana, whichincludes cannabinol and tetrahydrocannabinol.

The next group of drugs includes the vitamins such as A, B, e.g. B₁₂, C,D, E and K, folic acid, thiamine.

The next group of drugs is prostaglandins, which differ by the degreeand sites of hydroxylation and unsaturation.

The next group of drugs is antibiotics, which include penicillin,chloromycetin, actinomycetin, tetracycline, terramycin, theirmetabolites and derivatives.

The next group of drugs is the nucleosides and nucleotides, whichinclude ATP, NAD, FMN, adenosine, guanosine, thymidine, and cytidinewith their appropriate sugar and phosphate substituents.

The next group of drugs is miscellaneous individual drugs which includemethadone, meprobamate, serotonin, meperidine, amitriptyline,nortriptyline, lidocaine, procaineamide, acetylprocaineamide,propranolol, griseofulvin, valproic acid, butyrophenones,antihistamines, anticholinergic drugs, such as atropine, theirmetabolites and derivatives.

The next group of compounds is amino acids and small peptides whichinclude polyiodothyronines e.g. thyroxine, and triiodothyronine,oxytocin, ACTH, angiotensin, met- and leu-enkephalin their metabolitesand derivatives.

Metabolites related to diseased states include spermine, galactose,phenylpyruvic acid, and porphyrin Type 1.

The next group of drugs is aminoglycosides, such as gentamicin,kanamicin, tobramycin, and amikacin.

Among pesticides of interest are polyhalogenated biphenyls, phosphateesters, thiophosphates, carbamates, polyhalogenated sulfenamides, theirmetabolites and derivatives.

For receptor analytes, the molecular weights will generally range from10,000 to 2×10⁶, more usually from 10,000 to 10⁶. For immunoglobulins,IgA, IgG, IgE and IgM, the molecular weights will generally vary fromabout 160,000 to about 10⁶. Enzymes will normally range from about10,000 to 6,000,000 in molecular weight. Natural receptors vary widely,generally being at least about 15,000 molecular weight and may be 10⁶ orhigher molecular weight, including such materials as avidin, thyroxinebinding globulin, thyroxine binding prealbumin, transcortin, etc.

In many applications of the subject fluorescein derivatives, it will bedesirable to have the ligand bonded to a support, either directly,through the intermediacy of a ligand, or directly to the support, whilebound to a ligand.

A wide variety of supports may be employed. The particles or supportscan be derived from naturally occurring materials, naturally occurringmaterials which are synthetically modified and synthetic materials. Ofparticular interest are polysaccharides, particularly crosslinkedpolysaccharides, such as agarose, which is available as Sepharose,dextran, available as Sephadex and Sephacyl, cellulose, starch, and thelike. Other materials include polyacrylamides, polystyrene, polyvinylalcohol, copolymers of hydroxyethyl methacrylate and methylmethacrylate, silicones, glasses, available as Bioglas, nucleic acids,poly(amino acids), cells or the like. In addition to solid particles,liquid particles may also be employed having a lipophilic or amphiphilicmembrane, which serves to contain an internal fluid and define a space.Such particles include vesicles, cells and liposomes.

The particles may be porous or nonporous, swellable or nonswellable byaqueous or organic media, normally having a variety of functionalities,such as hydroxyl, amino or carboxy, either bonded directly to thebackbone or by means of a spacer arm, crosslinked or noncrosslinked,smooth or rough surface, or the like.

The porous particles may have a wide variety of cut off sizes, generallyvarying from about 10,000 to many million molecular weight, usually notexceeding 20 million molecular weight.

As already indicated, a wide variety of linking chains may be employedbetween the fluorescein compound and the ligand and/or support. Thechoice of linking group will vary widely, depending upon the availablefunctionalities or functionalities which may be readily introduced, thedesired length of the linking arm, the desirability of having thelinking arm provide for a particular environment, chemical property orphysical property, e.g. positively or negatively charged, solubilityenhancement, dipole effects, or the like.

The following table indicates a variety of linking groups which may beemployed for linking the fluorescein compound to the ligand:

                  TABLE II                                                        ______________________________________                                                                        Ligand/                                                                       support                                       Fluorescein                     function-                                     functionality                                                                             Linking group       ality                                         ______________________________________                                        CO.sub.2 H  (NHKCO).sub.k       NH.sub.2                                      CO.sub.2 H orSO.sub.3 H                                                                   (NHK'NH).sub.k' NHK'NH                                                                            CO.sub.2 H                                                NHK'N(CH.sub.2 CH.sub.2).sub.2 NK'NH                              SH                                                                                         ##STR4##           CO.sub.2 H                                    COCH.sub.2 halo                                                                            ##STR5##           CO.sub.2 H                                    NH.sub.2    COKCO               NH.sub.2                                      ______________________________________                                    

wherein:

K is alkylene of from 1 to 8, usually 1 to 6 carbon atoms, K' isalkylene of from 2 to 6, usually 2 to 4 carbon atoms, and k and k' are 1to 6, usually 1 to 4. It is understood that the above table is merelyillustrative of the more common linking groups, other linking groupsbeing available in special situations. For example, where phenolicgroups are present, such as tyrosyl, aryl diazonium functionalities maybe employed. Furthermore, it is understood that the functionalities forthe fluorescein and ligand or support may be reversed, with concomitantreversal of the direction of the linking group.

The subject compounds have many desirable properties. The products havesignificant water solubility which allow them to be conjugated to a widevariety of polypeptides, without significantly adversely affecting thewater solubility of the polypeptide, nor having the polypeptideadversely affect the spectroscopic properties of the subject compounds.

As for the spectroscopic properties of the compounds, the compoundsabsorb at relatively long wavelengths, generally in excess of 490 nm,more usually in excess of 510 nm. Thus, naturally occurring fluorescencewhich may be encountered when working with physiological fluids issubstantially avoided by employing exciting light at a wavelength rangewhich does not significantly excite the naturally occurring fluorescers.In addition, the compounds have relatively sharp absorption peaks andemission peaks. Because of this, efficient overlap can be obtainedbetween fluorescers and quenchers which allow for efficient quenching upto distances of about 70 A. The fluorescing compounds also have largeStokes shifts, so that the absorption band and emission band peaks areseparated by at least 10 nm, frequently by at least 15 nm. The largeStokes shifts minimize background interference with the observedfluorescence and permit more sensitive fluorescence detection byallowing the use of optical filters with high light transmittance.

The compounds of the subject invention are prepared in accordance withconventional means. The appropriate resorcinol and carboxylic acid oranhydride are combined in the presence of a Lewis acid e.g. zincchloride, and the mixture heated at an elevated temperature for asufficient time to provide the desired product. The product may then bepurified by conventional means.

The subject compounds find a wide variety of applications, particularlyfor use as conjugates to ligands and/or supports in protein bindingassays. The conjugates can be used for determining qualitatively,semi-quantitatively or quantitatively the presence of a compound ofinterest in a sample. Where the compounds are to be detected inphysiological fluids, the fluids may include serum, urine, saliva,cerebral spine fluid, lymph or the like. Where the compound of interestis involved in chemical processing or ecological concerns, the samplemay involve an aqueous medium, an organic medium, soil, inorganicmixtures, or the like.

For use in immunoassays or in other diagnostic situations, thespectroscopically active compounds of this invention will be conjugatedto a compound of interest, including a receptor for an analyte or aligand. (By receptor is intended any molecule which specifically bindsto a spatial and polar molecular organization, while a ligand is anorganic molecule having such organization.) The analyte will normally behaptenic or antigenic. Where these compounds do not have availablefunctionalities for linking, they will be modified to introduce such afunctionality, while still retaining the immunological properties in theresulting product. Those compounds which are analogs of the analyte,which analyte may also be referred to as a ligand, will be referred toas ligand analogs.

As indicated previously, the compounds of this invention may beconjugated to compounds which may be measured by known immunoassaytechniques. The resulting conjugates are reagents which complete in anassay medium for the compound of interest or analyte in a sample.Therefore, the conjugate retains a sufficient proportion of thestructure of the compound of interest to be able to complete with thecompound of interest for receptor, usually an antibody.

The analytes or their analogs, receptors or ligands, which areconjugated to the spectroscopically active compounds of this inventionare characterized by being monoepitopic or polyepitopic.

The assays will normally involve a change of spectroscopic propertiesdue to a change in environment about the spectroscopically activecompound or the bringing together of a fluorescer-quencher pair withinsufficient proximity for the quencher to interact with the fluorescer.Alternatively, methods can be employed which involve the separation ofassociated and unassociated fluorescer and the detection of thefluorescer in one or both of the fractions.

In a first assay, steric exclusion is involved, in that receptors orantibodies for the ligand and for the fluorescer are employed, wheresimultaneous binding of the receptor for the ligand and receptor for thefluorescer is inhibited. Furthermore, when the receptor for thefluorescer (antifluorescer) is bound to the fluorescer, the fluorescenceof the fluorescer is substantially diminished. Further reduction if notcomplete inhibition of fluorescence can be achieved by conjugation ofquencher to the antifluorescer. This assay is extensively described inU.S. Pat. No. 3,998,943, issued Dec. 21, 1976. The fluorescein which isemployed there may be substituted with the fluorescent compounds of thesubject invention. The assay is described in Columns 3-5 of the subjectpatent, which description is incorporated herein by reference.

Generally, the method involves combining the samples suspected ofcontaining the analyte, the conjugate of the ligand and fluorescer,anti-fluorescer, and receptor for ligand or antiligand, when ligand isthe analyte. The materials are combined in an aqueous medium at a pH inthe range of about 5 to 10, usually in the range of about 6 to 9, at atemperature in the range of about 10° to 45° C. and the fluorescencedetermined either as a rate or equilibrium mode, readings being takenwithin about 1 second to 1 hour after all materials have been combinedfor a rate mode, while for an equilibrium mode, readings may be takenfor as long as up to about 24 hours or longer.

In the next immunoassay technique, a fluorescer-quencher pair isemployed, where one of the members of the pair is conjugated to a memberof a specific binding pair, ligand and antiligand, and the otherchromophor member is bound to the same or different member of thespecific binding pair. For example, the fluorescer and the quencher maybe bound to different molecules of antiligand, so that when the twoconjugated antiligands are brought together with antigen, the fluorescerand quencher are brought within quenching distance. Alternatively, onecould bind one of the chromogens to the ligand and the other chromogento the antiligand. This assay is extensively described in U.S. Pat. No.3,996,345. The assay technique is described beginning with Col. 17 andending at Col. 23, which description is incorporated herein byreference. The ratios of chromogen to ligand and receptor is describedin Cols. 4-6, which description is incorporated herein by reference.

The assay is carried out in substantially the same manner as describedabove, except that in this assay, the fluorescer conjugates and quencherconjugates are added in conjunction with the sample and the fluorescencedetermined in comparison to an assay medium having a known amount of theanalyte.

Other techniques may also be employed with the subject compounds, suchas techniques involving heavy atom quenching as described in co-pendingapplication Ser. No. 824,576, filed Aug. 13, 1977, now abandoned, orother assay techniques where a fluorescent molecule is desired whichemits light at a wavelength substantially above the light emitted byfluorescent compounds naturally present in physiological fluids or othersamples to be analyzed.

Finally, the subject conjugates may be used in conjunction with supportsas described in U.S. Patent Application Ser. No. 964,099, filed November24, 1978 now U.S. Pat. No. 4,275,149. These assays are predicated uponhaving the fluorescer molecule available in bulk solution forinteraction with a single modulator or bound to a particle, where theparticle environment prevents the interaction. Alternatively, theparticle can provide an environment which modulates the fluorescentsignals when the fluorescer conjugate is bound to the particle.

EXPERIMENTAL

The following examples are offered by way of illustration and not by wayof limitation.

(All temperatures not otherwise indicated are centigrade. All parts andpercents not otherwise indicated are by weight, except for mixtures ofliquids which are by volume. The following abbreviations are employed:TLC-thin layer chromatography.

EXAMPLE I2-Methoxy-4,5,7-trichloro-9-(2'-carboxy)-6-hydroxy-3H-xanthen-3-one

A mixture of 2,4-dihydroxy-3,5-dichloro-2'-carboxy benzophenone (160 mg,0.05 mmole) and 2-chloro-4-methoxyresorcinol (87 mg, 0.05 mmole) washeated in an open test tube immersed in an oil bath at 190° for 10 min.The tube was cooled and the residue dissolved in 1 N sodium hydroxide (2ml), acidified to pH 1 with conc. HCl, filtered and dried. The crude dyewas dissolved in methanol and purified by TLC using acetic acid:ethylacetate:CH₂ Cl₂ (1:20:79). The orange fluorescent band R_(f) 0.5 wasisolated to give the product. The λ_(max) ^(abs) was 514 nm and theλ_(max) ^(em) was 541 nm in 0.05 M phosphate buffer, pH 8.0. The quantumyield was 0.8.

EXAMPLE II 2,4-dihydroxy-3,5-dichloro-2'-carboxybenzophenone

To a solution of 1 g of 2,4-dihydroxy-2'-carboxybenzophenone in 5 mlanhydrous ether, sulfuryl chloride (2 ml) was added dropwise and thesolution stirred for 2 hrs. at room temperature. After removing thesolvent on a Rotovap, the residue was dissolved in 10% sodium carbonate,acidified with conc. HCl and filtered. The solid was dissolved in about1 ml ethyl acetate and added dropwise to a rapidly stirring hexanesolution. The resulting precipitate was filtered and dried under vacuumto give the product (0.55 g). TLC:R_(f) 0.6, CH₂ Cl₂ :MeOH:AcOH(89.5:10:0.5). m.p. 207° C.-210° C.

EXAMPLE III 2,4-Dihydroxy-2',4' or 5'-dicarboxybenzophenone

A mixture of the m- or p-carboxy substituted fluorescein (8 g) was addedslowly to a hot (170° conc. sodium hydroxide solution (18 g sodiumhydroxide in 10 ml water) and maintained at that temperature for 1.5 hr.followed by cooling and diluting with 20 ml water. After acidifying topH 1 with conc. HCl, the resulting precipitate was filtered to give thedesired product TLC R_(f) 0.4, CH₂ Cl₂ :MeOH:AcOH (89.5:10:0.5).

EXAMPLE IV 2,4-Dihydroxy-5,3',6'-trichloro-2',4' or5'-dicarboxybenzophenone

To a rapidly stirring solution of 3,6-dichloro-4-carboxyphthalicanhydride (5 g in 50 ml tetrachloroethane) (prepared from3,6-dichlorobenzene-1,2,4-tricarboxylic acid by heating at 180° for 1hr.) was added 14 g aluminum chloride and 3.4 g 4-chlororesorcinol andthe mixture heated at 90° for 6 hrs. After quenching with ice and 1 NHCl, the black solution was extracted three times with ether, thecombined ether extracts dried over magnesium sulfate and the etherremoved in vacuo. The residue was dissolved in 5% sodium bicarbonate,the solution filtered and the basic solution extracted with ether (5×50ml) until the extracts were clear. The aqueous layer was acidified to pH1 with HCl and extracted with ether. The combined ether extracts weredried over magnesium sulfate and the ether evaporated to yield about 8 gof crude product. The crude product was purified by columnchromatography on 200 g silica gel (Merck 60) and eluted with aceticacid:acetone:benzene (2:32:66), thereby isolating a mixture of isomersR_(f) 0.4. The solid material was stirred with 1 N HCl overnight,filtered and dried to give 2 g of the product m.p. 203°-205° C.

Following the procedure described in Example I, except as otherwisedescribed, the following Table III indicates a number of additionalcompounds which were prepared and their properties.

                  TABLE III                                                       ______________________________________                                              Product                                                                      6-hydroxy-                                                                    3H--xanthen-                                                                  3-one      Resorcinol      Benzophenone                                  Ex.  (X)        (R)        mg   B         mg                                  ______________________________________                                        V    2-methoxy- 4-methoxy-R                                                                              68   2,4-dihydroxy-                                                                          164                                      5,7-dichloro-              3.5-dichloro-                                      2'-carboxy-X               2'-carboxy-B                                  VI   2-methyl-  4-methyl-R 70   2,4-dihydroxy-                                                                          150                                      2',4' or 5'-               2',4' or 5'                                        dicarboxy-X                dicarboxy-B                                   VII  2-methoxy- 4-methoxy-R                                                                              170  2,4-dihydroxy-                                                                          300                                      2',4' or 5'-               2',4' or 5'-                                       dicarboxy-X                dicarboxy-B                                   VIII 2-methoxy- 2-chloro-4-                                                                              170  2,4-dihydroxy-                                     4-chloro-  methoxy-R       2',4'- or 5'-                                      2',4'- or 5'-              dicarboxy-B                                        dicarboxy-X                                                              IX   2-methoxy-5,                                                                             4-methoxy-R     2,4-dihydroxy-                                     7-dichloro-                3,5-dichloro-                                      2',4' or 5'-               2',4'- or 5'-                                      dicarboxy-X                dicarboxy-B.sup.4                             X    2-methoxy- 4-methoxy-R                                                                              84   2,4-dihydroxy-                                                                          200                                      7,3',6'-                   5,3',6'-                                           trichloro-2',              trichloro-2',                                      4'- or 5'-                 4'- or 5'-                                         dicarboxy-X                dicarboxy-B                                   XI   2-methoxy- 2-chloro-  75   2,4-dihydroxy                                                                           130                                      4,5,7,3',6'-                                                                             4-methoxy-R     3,5,3',6'-                                         pentachloro-               tetrachloro-                                       2',4'- or 5'-              2',4'- or 5'-                                      dicarboxy-X                dicarboxy-B.sup.5                             XII  4-methoxy- 2-methoxy-R                                                                              140  2,4-dihydroxy-                                                                          300                                      2',4'- or 5'-              2',4'- or 5'-                                      dicarboxy-X                dicarboxy                                     ______________________________________                                        Reaction.sup.1        λmax (nm).sup.3                                                                    quantum                                     Ex.    Conditions  R.sub.f.sup.2                                                                        abs    em   yield                                   ______________________________________                                        V      a           0.2    505    535  0.78                                    VI      a.         0.6    496    520  0.9                                     VII    a           0.5    500    534  0.78                                    VIII   a                  505    534  0.88                                    IX      b.         0.3    505    535  0.78                                    X      b           0.4    517    541  0.91                                    XI     b           0.3    531    555  0.82                                    XII    c                  501    none                                         ______________________________________                                        .sup.1                                                                            a - Conditions of Example 1                                                   b - Conditions of Example 1 except adding 20 mg ZnCl.sub.2                    with temperature of 165°                                               c - Conditions of Example 1 except 30 min                                 .sup.2                                                                            Eluting systems are as follows:                                           Ex.      Solvent            Ratio                                             5        AcOH:EtOAc:CH.sub.2 Cl.sub.2                                                                     1:10:89                                           6        AcOH:MeOH:CH.sub.2 Cl.sub.2                                                                      1:15:84                                           7        AcOH:CH.sub.2 Cl.sub.2 :EtOAc                                                                    1:10:89                                           8        AcOH:MeOH:CH.sub.2 Cl.sub.2                                                                      1:15:85                                           9        AcOH:EtOAc:CH.sub.2 Cl.sub.2                                                                     1:20:79                                           10       AcOH:Acetone:Benzene                                                                             1:33:66                                           11       AcOH:EtOAc:CH.sub.2 Cl.sub.2                                                                     1:20:79                                           12       AcOH:MeOH:CH.sub.2 Cl.sub.2                                                                      1:15:85                                           .sup.3 Measurement taken in 0.05 M PO.sub.4.sup.-3, pH 8.0                    .sup.4 Prepared from an analogous fluorescein derivative in                   accordance with Example 3                                                     .sup.5 Prepared from 2,4-dihydroxy-5,3',6'-trichloro-2',4'- or                5'-dicarboxybenzophenene in accordance with Example 2                     

EXAMPLE XIII

A solution of 200 mg of the product of Example XI in 3 ml dry THFcontaining N,N'-dicyclohexyl carbodiimide and N-hydroxy succinimide (49mg) was stirred at room temperature overnight. Separately, t-butylglycinate hydrochloride (1 g) was dissolved in 4 ml DMF and 1 mltriethylamine added. The resulting solution was filtered and cooled toice-bath temperature (4°). To this was added the ester solution preparedabove and the reaction mixture stirred in the cold room overnight. Afterremoving the solvents in vacuo, the residue was stirred in hexane,filtered and dried. The crude dye was further purified by preparativeTLC using AcOH:EtOAc:CH₂ Cl₂ (1:20:79), R_(f) 0.7. The t-butyl ester washydrolysed by stirring in 2 ml trifluoroacetic acid for 2 hrs. Thevolatiles were removed in vacuo and the residue dissolved in 10 ml 1 Nsodium hydroxide, followed by acidification to pH 1 with HCl, theresulting precipitate filtered and dried in vacuo yielding 80 mg of theproduct. The spectroscopic properties are as follows: λ_(max) ^(abs),531; λ_(max) ^(em), 555, quantum yield 0.82 in 0.05 M phosphate buffer,pH 8.0.

EXAMPLE XIV

A mixture of 15 mg of a product of Example XIII, 6 mg ofN,N'-dicycloheyl carbodiimide and 3 mg of N-hydroxy succinimide in 1 mlTHF was stirred overnight. After filtering the solution, the THF wasremoved in vacuo and the residue triturated with hexane. The resultingsolid was filtered and washed with more hexane to yield the desiredester.

Varying ratios of the dye to protein were prepared by dissolving varyingamounts of the above ester in dry DMF and adding the solution to a humanIgG solution in 0.05 M phosphate buffer, pH 0.8. The reaction wasallowed to proceed overnight at 0°-5° C., followed by purification overa Sephadex G-25 column prepared in 0.05 M phosphate buffer, pH 8.0. Dyeto protein ratios were calculated from the UV and visible spectra bymeasuring absorption at 535 and 280 nm. The following indicates theratios and a plot of the quantum yields of the various conjugates vsdye/protein ratio.

                  TABLE IV                                                        ______________________________________                                        Dye/Protein   Fluorescence                                                    mole ratio    Quantum Yield                                                   ______________________________________                                        1.6           0.45                                                            2.1           0.46                                                            2.9           0.46                                                            "             0.41                                                            3.9           0.42                                                            4.1           0.45                                                            4.9           0.39                                                            5.2           0.32                                                            7.2           0.27                                                            9.6           0.17                                                            ______________________________________                                    

The subject invention provides novel compounds which have importantspectroscopic properties, providing for absorption at long wavelengths,high extinction coefficients, sharp absorption bands and fluorescentbands, and substantial spacing between absorption and fluorescencebands. These properties are particularly desirable and important to thedevelopment of fluorescent techniques for the detection of a widevariety of materials. In addition, members of the subject genus can alsobe used as quenchers where there is a 4-oxy substituent, in having no orsubstantially no fluorescence emission while absorbing light atwavelengths to provide for quenching of fluorescence by a fluorescer inthe excited state in close proximity to the quencher.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A compound of the formula ##STR6## wherein: oneof the γs will be of the formula --(O).sub.ε ρ and the other isδ;wherein ε is 0 or 1 and ρ is an aliphatic group of from about 1 to 12carbon atoms and 0-1 carboxyl groups; the δ's are the same or differentand are hydrogen or halogen of atomic number 17 to 80; L is a bond ordivalent organic radical of up to 20 carbon atoms; α may be takentogether with Ψ to define an active group capable of forming a stablecovalent bond with carbon, nitrogen or oxygen or is an organic compound,and is at other than the ortho position when L is benzene and Ψ and αare taken together to define carboxy; Ψ, when not taken together with αis a linking group to α; v is 1 when Ψ is taken together with α and isotherwise on the average at least 1 and not more than the molecularweight of α divided by
 500. with the proviso that when ε is 0, one ofthe δ's may be an aliphatic group of from about 1 to 12 carbon atomsasymmetrically substituted in relation to ρ.
 2. A compound according toclaim 1 conjugated to a support.
 3. A compound of the formula ##STR7##wherein: one of the Gs is of the formula --(O)_(e) R and is otherwise D,where e is 0 or 1 and R is an aliphatic group of from 1 to 6 carbonatoms having from 0 to 1 carboxy groups;Z is carboxy or sulphonoxy; D ishydrogen or halogen of atom number 17 to 80; W is a bond or divalentorganic radical having up to 10 carbon atoms; Y may be taken togetherwith A to form an active functionality capable of forming a covalentbond with carbon, nitrogen or oxygen when not taken together with A, Yis a linking functionality; A when not taken together with Y is a ligandor receptor of at least about 125 daltons; X is halo of atomic number 9to 80; m is 1 to 3; p is 0 to 3, with the sum of m and p being notgreater than 4;n will be 1 when Y and A are taken together or otherwisebe on the average 1 to the molecular weight of A divided by 500, withthe proviso that when W is a bond and Y and A are taken together todefine carboxy, Y is at other than the ortho position.
 4. A compoundaccording to claim 3, wherein e is
 0. 5. A compound according to claim3, wherein e is
 1. 6. A compound of the formula ##STR8## wherein: one ofG' or G" is --(O)_(e') R' and is otherwise D', wherein e' is 0 or 1 andR' is alkyl of from 1 to 3 carbon atoms;D' is hydrogen or halogen ofatomic number 17 to 80; Y' may be taken together with A' to define anon-oxo-carbonyl group and is otherwise a non-oxo-carbonyl group formingan amide group with an amino group of A'; A' when not taken togetherwith Y', is a ligand or receptor of at least about 125 daltons; W' is abond or aliphatic linking group of from 1 to 12 atoms other thanhydrogen, which are carbon, nitrogen, oxygen or sulfur, there being from0 to 8 carbon atoms and 0 to 8 heteroatoms, wherein nitrogen is presentas amino or amido; and oxygen and sulfur are present as oxy, oxo or thesulfur analogs thereof; Z' is carboxy; X' is halo of atomic number 9 to80; n' is 1 when Y' and A' are taken together and is otherwise on theaverage at least 1 to the molecular weight of A' divided by about 1,000;m' is 0 to 2; p' is 0 to 3, the sum of m' and p' being not greater than4, with the proviso that, when W' is a bond and Y' and A' are takentogether to define carboxy, Y' is at other than the ortho position.
 7. Acompound according to claim 6, wherein A' is taken together with Y'. 8.A compound according to claims 6 or 7, wherein R' is methyl.
 9. Acompound according to claims 6 or 7, wherein at least one of the D's andX's are chloro.
 10. A compound according to claim 6, wherein A' is aligand.
 11. A compound according to claim 6, wherein A' is a receptor.12. A compound of the formula ##STR9## wherein: D' is hydrogen orchloro;e' is 0 or 1; the sum of m' and p' is 0 to 4, wherein m' is 0 to2; A' is hydroxyl or a ligand or receptor of at least about 125 daltons;and --COA' is at other than the ortho position.
 13. A compound accordingto claim 12, wherein e' is
 0. 14. A compound according to claim 12,wherein e' is
 1. 15. A compound according to claims 13 and 14, whereinA' is hydroxyl.
 16. A compound according to claims 13 and 14, wherein A'is a ligand.
 17. A compound of the formula ##STR10## wherein: e' is 0 or1;m' is 0 to 2; p' is 0 to 3, the sum of m' and p' being not greaterthan 4; D' is hydrogen or chloro; and A' is hydroxyl or a ligand orreceptor of at least about 125 daltons; with the proviso that, when A'is hydroxyl, --COA' is at other than the ortho position.
 18. A compoundaccording to claim 17, wherein e' is
 0. 19. A compound according toclaim 17, wherein e' is
 1. 20. A compound according to claims 17 and 18,wherein A' is hydroxyl.
 21. A compound according to claims 13 or 17,bound to a support.
 22. A compound according to claims 18 or 19, whereinA' is a ligand.